Luminescence is a phenomenon in which energy is specifically channeled to a molecule to produce an excited state. Return to a lower energy state is accompanied by release of a photon. Luminescence includes fluorescence, phosphorescence, chemiluminescence and bioluminescence. Bioluminescence is the process by which living organisms emit light that is visible to other organisms. Where the luminescence is bioluminescence, creation of the excited state derives from an enzyme catalyzed reaction. Luminescence can be used in the analysis of biological interactions.
Interactions between proteins and other molecules play a key regulatory role in almost every biological process. For example, cytosolic and cell surface protein-protein interactions play major roles in normal cellular functions and biological responses. In particular, many cytosolic and cell surface protein-protein interactions are involved in disease pathways. For example, attacks by pathogens such as viruses and bacteria on mammalian cells typically begin with interactions between viral or bacterial proteins and mammalian cell surface proteins. In addition, many protein-protein interactions between factors in cellular transcriptional machineries are also valuable drug targets. Protein-protein interactions are also involved, for example, in the assembly of enzyme subunits; in antigen-antibody reactions; in forming the supramolecular structures of ribosomes, filaments, and viruses; in transport; and in the interaction of receptors on a cell with growth factors and hormones. Products of oncogenes can give rise to neoplastic transformation through protein-protein interactions. Thus, many techniques have been developed to identify and characterize these interactions.
One technique for assessing protein-protein interaction is based on fluorescence resonance energy transfer (FRET). In this process, one fluorophore (the “donor”) transfers its excited-state energy to another fluorophore (the “acceptor”), which usually emits fluorescence of a different color. According to Forster equation (Forster, T., (1948) Ann. Physik., 2, 55 and Forster, T., (1960) Rad. Res. Suppl., 2, 326), FRET efficiency depends on five parameters: (i) the overlap between the absorption spectrum of the second fluorophore and the emission spectrum of the first fluorophore, (ii) the relative orientation between the emission dipole of the donor and the absorption dipole of the acceptor, (iii) the distance between the fluorophores, (iv) the quantum yield of the donor, and (v) the extinction coefficient of the acceptor.
FRET has been used to assay protein-protein proximity in vitro and in vivo by chemically attaching fluorophores such as fluorescein and rhodamine to pairs of purified proteins and measuring fluorescence spectra of protein mixtures or cells that were microinjected with the labeled proteins (Adams et al, (1991) Nature, 349, 694-697).
The cloning and expression of Green Fluorescent Protein (GFP) in heterologous systems opened the possibility of genetic attachment of fluorophores to proteins. In addition, the availability of GFP mutants with altered wavelengths (Heim et al., (1994) Proc. Natl. Acad. Sci. USA., 91, 12501-12504) allowed their use as FRET pairs.
An attractive application allowed by GFP-based FRET is the in vivo assay of protein interactions in organisms other than yeast. For example, fusion of GFP and BFP to the mammalian transcriptional factor Pit-1 showed homo-dimerization of Pit-1 in live HeLa cells (Periasamy, A. and Day, R. N., (1998) J. Biomed. Opt., 3, 1-7). In this type of assay, interactions can be examined in the proteins' native organism, such that cell-type specific modifications and/or compartmentalization of the proteins are preserved. Additionally, compartmentalization of these interacting proteins is potentially visible in the microscope.
FRET, however, has several limitations. As with any fluorescence technique, photobleaching of the fluorophore and autofluorescence of the cells/tissue can significantly restrict the usefulness of FRET, and, in highly autofluorescent tissues, FRET is essentially unusable. Also, if the excitation light easily damages the tissue, the technique may be unable to give a value for healthy cells. Finally, if the cells/tissues to be tested are photoresponsive (e.g., retina), FRET may be impractical because as soon as a measurement is taken, the photoresponse may be triggered.